FHIT Gene Therapy in Cancer Prevention and Treatment. We have developed murine upper digestive tract cancer models induced by oral /V-nitrosomethylbenzylamine(NMBA) or 4-nitroquinoline 1-oxide (NQO) treatment. Wild type (WT) mice are not very susceptible to these carcinogens but mice deficient for either Fhit or p53 develop a tumor burden up to 10 times greater than WT mice on exposure to NMBA or NQO, on predictable schedules. Mouse forestomach tumor burden is dramatically reduced by FHIT therapy early (tumor prevention) or late (tumor regression) after carcinogen exposure, and lung and cervical cancer studies are in progress. A caveat to mouse preclinical models is the prevailing notion that mouse tumors exhibit less genetic complexity and heterogeneity than human counterparts, so that human cancers may be less responsive to FHIT gene therapy. The proposed study aims to address this concern by testing FHIT gene therapy in genetically complex mouse tumors in the recombinant mouse cross, Fhit+/-xTrp53+/-, with induced forestomach and oral cancers, to show that Fhit, as a gatekeeper gene product whose loss initiates the neoplastic process, can prevent or reverse tumors after AAVFHIT delivery The FHIT locus is exquisitely susceptible to replication damage on exposure to genotoxic agents and Fhit protein is lost or reduced early in development of precancerous lesions of upper aerodigestive tract tumors. Research in this Project is based on the hypotheses that replacement of FHIT in these lesions could: a) eradicate the altered cells in the "cancer field" of these organs, thus preventing recurrences; b) reverse progression of established cancers; c) allow identification of pathways altered by Fhit loss during development of preneoplasia in Fhit deficient animals, before and after FHIT gene therapy, and of protein targets for pharmacological reactivation of Fhit signal pathways. Thus the aims of this research project are to: 1) prevent and reverse preneoplastic and neoplastic lesions, respectively, in forestomachs of Fhit+/- and Fhit+/-p53+/-mice by FHIT gene therapy; 2) optimize the protocol for NQO induction of oral cancers in the tumor suppressor deficient mice and prevent and reverse preneoplasias and neoplasias of the oral cavity in Fhit+/- and Fhit+/-p53+/-mice by FHIT gene therapy; 3) "cure" the Fhit and Fhit/p53 deficient mice of NMBA and NQO-induced lesions by multiple FHIT gene therapy doses or FHIT gene therapy plus Fhit pathway targeted drug treatment. In each specific aim Fhit-/- mice will be included and tissues from mice with and without FHIT gene therapy will be assessed for expression of cell cycle, DNA damage response and apoptosis-associated proteins, as well as Fhit- interacting proteins to identify the signal pathways altered by Fhit absence, restored by Fhit replacement, and likely to serve as drug targets for treatment of upper digestive tract and other cancers.